The reliability of gene expression studies by mRNA quantification is highly dependent upon several experimental procedures, including the choice of reference genes used for data normalization. In order to contribute to gene expression studies in mesial temporal lobe epilepsy (MTLE) we used microarray data, followed by real time quantitative PCR validation of selected housekeeping genes, to determine the most appropriate reference genes to be used in human hippocampal tissue gene expression studies. Our results unequivocally showed a significant impact of the reference gene chosen for normalization on the overall results of expression studies, clearly demonstrating the importance of adequate validation using stable reference genes. In addition, we found that HPRT, NSE, SDHA and SYP are suitable genes to be used as reference for normalization in expression studies of hippocampal tissue obtained from patients with MTLE.